Transglutaminases are a family of calcium-dependent enzymes mediating covalent cross-linking reactions between specific peptide bound γ-glutamyl residues and various primary amino groups of aliphatic amines, lysines or polyamines, acting as amine donor substrates (Davies, et al., Adv. Exp. Med Biol. 250, 391-401, 1988). These enzymes stabilize biological structures via the formation of isopeptide cross-links. In mammals, at least five enzymatically active transglutaminases have been identified, cloned and sequenced. The number of proteins acting as glutaminyl substrates for transglutaminases is restricted, and no obvious consensus sequence around these substrates' glutamines has been found.
Three main lines of investigation have been conducted surrounding transglutaminases. These enzymes have been used to label membrane proteins and, in the absence of exogenous amines, to catalyze the formation of (γ-glutamine)-lysyl cross-links between them. The labeling is quite specific and can be carried out under mild (physiological) reaction conditions. Thus, for example, transglutaminases were used to study rhodopsin in the intact disc membrane, as only residues of rhodopsin located in the aqueous phase in the exposed side of the disc membranes were expected to be labeled. In these experiments, rhodopsin was labeled by transglutaminase using putrescine and dansylcadaverine as detectable substrates.
The role of transglutaminases in living cells also has been studied, for example, using the cell-penetrating labeled substrate fluoresceincadaverine for detecting amine acceptor protein substrates accessible to active transglutaminase in living cells. A similar strategy was employed using 5-(biotinamido)-pentylamine as a label. Such labeled substrates can be detected directly, for example by fluorescence, or can be detected indirectly, for example using antibodies, to identify native proteins to which the labeled substrate has been covalently attached by transglutaminase. See, Pober, J. S. et al., Biochemistry, Vol. 17, No. 11:2163-2169 (1978); Lajemi, M. et al., Histochemical Journal 29:593-606 (1997).
More recently, an investigation was carried out to determine if polyglutamine is a transglutaminase substrate. It was determined that as long as polypeptides including stretches of polyglutamine are rendered sufficiently soluble by the flanking residues, all were excellent substrates of transglutaminase. Based upon these studies, it was speculated that certain diseases such as Spinocerebellar ataxia Type I, Machado-Joseph disease, and Dentato-Rubral pallidoluysian atrophy which are characterized by proteins having polyglutamine stretches, may arise as a result of aggregation of such proteins acted upon by a transglutaminase.
It also is described in U.S. Pat. No. 5,525,336 (the disclosure of which is incorporated herein by reference in its entirety) that transglutaminase and corneocyte proteins, the natural substrates of transglutaminases, can be used together as cosmetic treatments to cross-link preparations of corneocyte proteins to the outer layer of skin, hair or nails to form a protective layer on the skin, hair or nails.
U.S. Pat. No. 5,490,980 describes selecting agents having or modifying agents to have an aliphatic amine, and then attaching those agents to skin, hair or nails using transglutaminase. While the idea was sound in principle, in practice the '980 applicants achieved results that were barely above background. (See Example Section of '980 patent). An aliphatic amine was applied in the examples as a single linking molecule or prophetically in clusters (according to a formula in the '980 patent). In selecting the amine moiety of the pair of known transglutaminase substrate moieties, the '980 patent taught away from using the carboxamide substrate moiety.